Throughout this specification, including any claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps.
It must be noted that, as used in the specification and any appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.
Ranges are often expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment.
Chemotherapy
In general, cancer treatment by chemotherapy is limited by the need to deliver a high concentration of the anti-cancer drug selectively to the malignant cells. As a consequence, many methods for the efficient and selective delivery of a drug to the targeted malignant cells have been developed.
Many such methods employ prodrugs, which may be described, generally, as pharmacologically inactive (or relatively inactive) chemical derivatives of a drug molecule that require a transformation within the body in order to release the active drug.
In one approach, known as enzyme prodrug therapy (EPT), the transformation is effected by a particular enzyme, for example, carboxypeptidase G2 (CPG2) or nitroreductase (NR). Examples of such therapies include antibody directed enzyme prodrug therapy (ADEPT) and gene directed enzyme prodrug therapy (GDEPT), briefly described below. Other enzyme prodrug therapies include ligand-directed enzyme prodrug therapy (LIDEPT) (see, e.g., Springer and Marais, 1997) and bacteria directed enzyme prodrug therapy (BDEPT) (see, e.g., Satchi and Duncan, 1998). See also, for example, Kirn, 2000.
ADEPT
The ADEPT approach separates the targeting from the cytotoxic functions in a two-step treatment. The selective component is an antibody (Ab) within an enzyme conjugate. The Ab binds antigen preferentially expressed on the surface of tumour cells. In the first step, the Ab-enzyme conjugate is administered and time is allowed for it to accumulate at the tumour and to clear from blood and normal tissues. In the second step, a non-toxic prodrug is administered that is converted specifically by the enzyme at the tumour into a low molecular weight toxic drug. The interstitial tumour transport of these low molecular weight cytotoxic agents thus generated is more favoured than those of large immuno-conjugates such as immunotoxins. This allows greater tumour access for the toxic component. An amplification feature is inherent in ADEPT whereby one Ab-enzyme conjugate molecule can catalyse the conversion of many molecules of the prodrug into the cytotoxic drug, enabling higher concentrations of drug at the tumour than one-step Ab delivery systems. Another important factor is the by-stander effect, which effects killing of surrounding tumour cells even though they do not express tumour antigen or do not bind Ab-enzyme conjugate. The main drawback currently remains the immunogenicity of the Ab-enzyme conjugates which precludes the administration of repeated doses of the conjugate.
A number of papers review in detail the main features of ADEPT systems, including: Senter et al., 1993; Bagshawe et al., 1994; Deonarain and Epenetos, 1994; Jungheim and Shepherd, 1994; Niculescu-Duvaz and Springer, 1995, 1996; Springer and Niculescu-Duvaz, 1995; Springer et al., 1995a; Hay and Denny, 1996; Melton and Sherwood, 1996.
GDEPT
Gene therapy for cancer may be defined broadly as a genetic technology aimed at modifying either malignant or non-malignant cells for therapeutic gain. “Suicide” gene therapy approaches include GDEPT and VDEPT (virally directed enzyme prodrug therapy) (see, for example, Huber et al., 1995), the only difference between these approaches being that the former involves both viral and non-viral vectors.
Like ADEPT, GDEPT is a two-step treatment for tumours. Foreign enzymes are delivered to, and expressed in, target cells where they can activate subsequently administered non-toxic prodrugs to form active drugs. In the first step, a gene expressing the foreign enzyme is delivered. In the second step, a prodrug is administered that can be activated to form a toxic drug by the enzyme that has been expressed in the tumour. The foreign enzyme gene should be expressed exclusively, or with a relatively high ratio, in tumour cells compared with normal tissues and blood, and should achieve a sufficient concentration for clinical benefit. After gene delivery, prodrug administration must be delayed to permit protein expression in the targeted cells. The catalytic activity of the expressed protein should be sufficient for activation of the prodrug. Since expression of the foreign enzymes will not occur in all cells of a targeted tumour in vivo, a bystander cytotoxic effect is beneficial, whereby the prodrug is cleaved to an active drug that kills not only tumour cells but also neighbouring non-expressing tumour cells. This means that expression in less than 100% of tumour cells can still result in killing of all tumour cells. The foreign enzyme is usually expressed intracellularly, but by expressing the activating enzyme tethered to the outer cell surface of mammalian cells, potential advantages for GDEPT prodrug design are realized. The potential advantages of extracellular expression are twofold. Firstly, it should give an improved by-stander effect because the drug will be generated in the interstitial spaces within the tumour, rather than inside as with an intracellularly expressed activating enzyme. Secondly, the prodrug cannot enter cells to become activated and therefore non-cell-permeable prodrugs can be used. Thus, prodrugs which release drugs with intracellular targets may be rendered non-toxic by preventing their entry into cells. Upon activation, a potent and cell-permeable active moiety is released. This has already been demonstrated to be beneficial for prodrug-impermeable tumour cells (Marais et al., 1997). However, the potential for increased toxicity due to the diffusion of the active drug away from the tumour is a potential disadvantage, although this could also happen to active drugs from an intracellularly expressed enzyme.
A number of recent reviews cover the GDEPT approach, including: Zhang et al., 1995; Niculescu-Duvaz and Springer, 1997; Roth and Cristiano, 1997; Denny and Wilson, 1998; Encell and Loeb, 1998; Niculescu-Duvaz et al., 1998a, 1999; Springer and Niculescu-Duvaz, 1999a. Additional aspects are described in Springer and Marais, 1996a, 1996b.
Carboxypeptidase G2 (CPG2)
Peptidases are a class of enzymes (E) which act upon a substrate to cleave an amide linkage (—NH—C(═O)—) to give, usually, amino (—NH2) and carboxylic acid (—C(═O)OH) products.

One peptidase of particular interest is carboxypeptidase G2 (referred to herein as “CPG2”). CPG2 is a bacterial enzyme isolated from Pseudomonas R16 (Sherwood et al., 1985). It is a zinc-dependent metallo-proteinase which exists as a homodimer molecule (2×41,800 Da) containing two Zn2+ ions in each monomeric unit (Minton et al., 1984). The enzyme belongs to the group of calcium-binding zinc-endopeptidases from bacteria which contain thermolysin and other neutral peptidases from Bacillus subtilis and Aeromonas proteolytica (Matthews, 1988; Roswell et al., 1997).
CPG2 was first proposed by Bagshawe et al., 1988, and catalyses the scission of amidic (Springer et at., 1990a), urethanic or ureidic (Springer et al., 1995b; Dowell et al., 1996), bonds between a benzene nucleus and L-glutamic acid.
A preferred substrate for CPG2 is an L-glutamic acid group, linked to an aromatic ring via an amidic, carbamic, or ureidic linkage.

However, glutamic acid analogs are also acceptable substrates. For example, L-glutamic acid modified at the γ-carbon (e.g., with an amide, —CONH2, instead of an acid, —COOH) also serves as a suitable substrate for CPG2.
CPG2 is also tolerant as to whether the amide group is naked, or is part of a larger linkage, for example, a carbamate or a urea linkage.

For these compounds, CPG2 yields CO2, L-glutamic acid, and R-ZH, wherein when Z is —O— (carbamates), R-ZH is a hydroxyl compound, R—OH, and when Z is —NH— (ureas), R-ZH is an amino compound, R—NH2, where R is preferably an aromatic group.
CPG2 Activated Self-Immolative Prodrugs
A “self-immolative prodrug” can be defined as a compound which, following an activation process, generates an unstable intermediate that releases the active drug in a number of subsequent steps.
Typically: (i) the activation process is of an enzymatic nature and is distinct from the extrusion step; (ii) the drug is generated by an extrusion process, following the fragmentation of the prodrug; (iii) the site of activation will normally be separated from the site of extrusion.
Potential advantages of self-immolative prodrugs include: the possibility of altering the lipophilicity of the prodrugs with minimal effect on the activation kinetics; the improvement of unfavourable kinetics of activation due to unsuitable electronic or steric features; the range of drugs which can be converted to prodrugs is greatly extended and is unrestricted by the structural substrate requirements for a given enzyme.
In one class of CPG2 activated self-immolative prodrugs, shown below, the L-glutamic acid and the active drug are separated by a 4-hydroxy (where Z is —O—) or 4amino where Z is —NH—) substituted benzylic spacer.

The activation of these prodrugs involves two steps:                (i) the cleavage of the oxycarbonyl- or carbamoyl-L-glutamyl linkage by CPG2, followed by the spontaneous decomposition of the carbonic or carbamic acid thus formed with loss of CO2;        (ii) the fragmentation of the self-immolative intermediate by a 1,6-elimination mechanism (Wakselman, 1983), releasing a carbonic or carbamic acid which upon loss of CO2 generates an active drug (HNR2).        

In this way, the prodrug, upon self-immolation, releases an amine drug (and CO2) from a carbamate linkage. Other classes includes compounds which, upon self-immolation, release an aryl alcohol from an aryl ether; an aryl carboxylic acid from an aryl ester; an aryl alcohol (and CO2) from an aryl carbonate; and the like. Similar self-immolative prodrugs are described, for example, in Springer et al, 1995c, 1995d.
Nitrogen Mustards
Nitrogen mustards are related to sulfur mustard, (ClCH2CH2)2S, the “mustard gas” used during the First World War. Nitrogen mustards have the general formula (ClCH2CH2)2NR. In vivo, each 2-chloroethyl side-chain undergoes an intramolecular cyclisation with the release of a chloride ion. The resulting highly reactive ethylene immonium derivative can interact with DNA and other molecules, for example, as an alkylating and/or crosslinking agent. Nitrogen mustards are useful, for example, in the treatment of proliferative conditions, such as cancer.
Nitrogen mustard analogues, in which the chloro group is replaced by other groups, such as other halogens (e.g., bromo, iodo) and other good leaving groups (e.g., sulfonates, such as mesyloxy, —OSO2Me) are also known, and are included in the class denoted “nitrogen mustards.”
Nitrogen mustards may conveniently be grouped according to the group R. For example, two groups are phenolic nitrogen mustards and anilinic nitrogen mustards.
CPG2 Activated Nitrogen Mustard Prodrugs
The EPT approach has been applied to nitrogen mustard drugs. For example, in one approach, CPG2 acts upon the prodrug to yield a drug, R-ZH, which is a phenolic (Z is —O—) or anilinic (Z is —NH—) nitrogen mustard compound.

Various nitrogen mustard prodrugs are described, for example, in Springer 1990b, 1991, 1994, 2000, and 2002.
CPG2 Activated Nitrogen Mustard Self-Immolative Prodrugs
The CPG2 activated self-immolative prodrug approach, discussed above, has also been applied to nitrogen mustards. In one approach, the drug, NHR2, is an anilinic nitrogen mustard compound. The prodrug is activated by CPG2, undergoes self-immolation, and releases the anilinic nitrogen mustard.

Springer et al, 1996, describe a number of nitrogen mustard prodrugs, including compounds of the following structure (see, for example, compounds 19 and 20 on pages 22 and 23 and in FIG. 3, therein).

In each case, the nitrogen atom (indicated by the arrow, above) of the carbamate group which is between the benzyl group of the self-immolative core and the phenyl group of the nitrogen mustard is unsubstituted, that is, the carbamate group is —O—C(═O)—NH—. This nitrogen atom, identified as Z1 therein, is soley described as —O— or —NH— (see page 3, line 5; page 6, line 24; page 7, line 1, page 7, line 7; page 11, line 1; and page 15 line 6, therein). Nowhere in this document is there provided any teaching or suggestion whatsoever that, as an alternative, the nitrogen atom of this carbamate group might be substituted.
The inventors have discovered that, surprisingly and unexpectedly, corresponding compounds, in which the nitrogen atom is substitued, for example, with a C1-7alkyl group, offer one or more pharmacological advantages, including but not limited to: (a) improved activity; (b) improved selectivity (e.g., against tumour cells versus normal cells); (c) reduction in required dosage amounts; (d) reduction in required frequency of administration; (e) reduced intensity of undesired side-effects; (f) fewer undesired side-effects.